Site-specific proteolysis plays a significant role in the regulation of basic cellular processes such as transcription, apoptosis, development, cell cycle, and signaling. Site specific proteolysis also plays critical roles in the lifecycle of viruses and other pathogenic organisms. Previously a genetic screen was developed for the isolation and characterization of site-specific proteases. This system demonstrated a high level of specificity in the ability to isolate and characterize a site-specific protease of relatively low abundance and activity from a complex cDNA pool. For the human immunodeficiency virus (HIV) the encoded protease is required for the replication of the virus and has been the target of novel anti-viral therapeutics. However, arising inhibitor resistant viral strains have become an increasingly significant clinical problem. Using the developed genetic selection, a bank of inhibitor-resistant mutants in this protease have been isolated including mutations which correlate with resistant clinical isolates. The rapid selection of such mutations has implications for the prediction of relevant mutations and may be applicable to other viral systems.